Defective Integrator activity shapes the transcriptome of patients with multiple sclerosis

High-depth RNA sequencing reveals down-regulation of HP1α/CBX5 in monocytes of multiple sclerosis patients, associated with major transcriptomic changes and reduced activity of the Integrator complex.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): The manuscript by Porozhan et al. entitled "Defective Integrator activity shapes the transcriptome of patients with multiple sclerosis" attempts at dissecting the role of the transcription factor CBX5 in the context of autoimmune demyelination.The authors identified by RNA-seq a sub-group of multiple sclerosis (MS) patients characterized by low levels of CBX5 in monocytes.Further analysis connected low CBX5 expression with extensive RNA dysmetabolism.Lastly, the authors were able to recapitulate many of the phenotypes observed in MS patients in CBX5 deficient mice with the MS model experimental autoimmune encephalomyelitis (EAE).This work is relevant to the MS research field as it sheds light on a previously unknown molecular mechanism underlying MS etiology.The study does a good job in combining multiple approaches.The experiments appear well executed and the results generally support the conclusions.However, several technical and conceptual limitations decrease the robustness and impact of the work.For these reasons, the following points need to be carefully addressed before I can endorse the manuscript for publication: -The observation of a subset of MS patients with low CBX5 levels needs to be independently replicated to exclude possible random effects due to the small sample size of the tested cohort and possible influence of confounding factors.For instance, the high-CBX5 group is enriched in CIS patients (likely treatment naïve) while the low-CBX5 group consists only in patients with clinically defined MS (likely under treatment).
-There is no rationale for the initial choice to profile monocytes in MS patients.Also, data on the purity of the isolated monocytes should be included as supplementary files.
-Although the symptomatic control group is an interesting choice, the initial RNA-seq screening requires an additional control group encompassing healthy subjects which will serve as real baseline for all the other experimental groups.
-The differences in the levels of CBX5 detected by RNA-seq need to be independently confirmed by qRT-PCR and western blotting.
-The plot of EAE clinical scores in Fig. 4B lacks the necessary statistics to confirm the significance of the differences between genotypes.
-The characterization of the EAE model is largely incomplete.The analysis of EAE scores should be complemented with histological evaluation of immune cell infiltration and demyelination in the spinal cord.In addition, the peripheral autoimmune response should be assessed in terms of pro-inflammatory vs tolerogenic mediators (Th1/Th17 T cells, cytokines and so on).
-It is not clear the number of mice that was used for the EAE experiment for each genotype.Same for the number of independent immunizations that were carried out.Given the high degree of variability of the model and the relatively small differences in clinical scores between genotypes, multiple immunizations are required to confirm the results.
-It is not clear the rationale for performing RNA-seq on CD4+ T cells in EAE mice while monocytes were investigated in MS patients.The analysis in EAE mice should be extended to monocytes for consistency.
-More than 200 loci have been found associated with MS risk in a recent GWAS (IMSGC, Science 2019).It would be relevant to test whether the low-CBX5 group carries any of those risk SNPs in the CBX5 locus.The manuscript by Porozhan et al. entitled "Defective Integrator activity shapes the transcriptome of patients with multiple sclerosis" attempts at dissecting the role of the transcription factor CBX5 in the context of autoimmune demyelination.The authors identified by RNA-seq a sub-group of multiple sclerosis (MS) patients characterized by low levels of CBX5 in monocytes.Further analysis connected low CBX5 expression with extensive RNA dysmetabolism.Lastly, the authors were able to recapitulate many of the phenotypes observed in MS patients in CBX5 deficient mice with the MS model experimental autoimmune encephalomyelitis (EAE).This work is relevant to the MS research field as it sheds light on a previously unknown molecular mechanism underlying MS etiology.The study does a good job in combining multiple approaches.The experiments appear well executed and the results generally support the conclusions.We thank the Reviewer for recognizing the relevance of our work for MS research and for his positive comments on our approaches and execution.
However, several technical and conceptual limitations decrease the robustness and impact of the work.For these reasons, the following points need to be carefully addressed before I can endorse the manuscript for publication: -The observation of a subset of MS patients with low CBX5 levels needs to be independently replicated to exclude possible random effects due to the small sample size of the tested cohort and possible influence of confounding factors.For instance, the high-CBX5 group is enriched in CIS patients (likely treatment naïve) while the low-CBX5 group consists only in patients with clinically defined MS (likely under treatment).To increase the robustness of our findings, we now analyze a publicly available RNA-seq data set from MS patient T cells.This analysis confirms that between patients with respectively low and high CBX5 expression, the genes that are differentially expressed are those highly dependent on promoter escape for their regulation.These data are now presented in new Sup.Figures 2A-2D.Regarding the enrichment of the high-CBX5 and low-CBX5 groups in different categories of patients, we agree that the sample size is small and that the diagram Fig1D only indicates a trend.This is now clearly addressed in the "discussion" section.
-There is no rationale for the initial choice to profile monocytes in MS patients.Also, data on the purity of the isolated monocytes should be included as supplementary files.Monocytes were chosen downstream of initial study by Carstensen et al, 2018 (Ref #18) showing increased levels of epitopes originating from endogenous retroviruses in monocytes from MS patients.These observations had suggested that monocytes would be a good tissue for the detection of unusual transcripts.This is now clearly stated in the revised version.Purity of the monocytes was verified by flow cytometry on patient #129, with the same approach as in Ref #18.This is now clearly stated in the "Material & Method" section, but unfortunately, we have not kept the .fcsfiles required to make the requested sup.figure.
-Although the symptomatic control group is an interesting choice, the initial RNA-seq screening requires an additional control group encompassing healthy subjects which will serve as real baseline for all the other experimental groups.Concerning the use of symptomatic controls, we believe that we have lacked clarity in the initial version.SCs are healthy, and do not have any disease.They have experience paresthesia without change in sensation, blurry vision explained by a lack of adjustment in vision, dizziness due to increased muscle tension, reduced muscle strength due to decreased physical activity and condition, etc.Thus, they have neurological symptoms, but no objective clinical or paraclinical findings define a specific neurological disease.In combination with MRI showing normal or unspecific findings, the designation of SC is determined through exclusionary criteria and, clearly, does not represent early MS.Finally, because they have been through an MS diagnosis process, we have good evidence for a normal neurological examination and MRI of the central nervous system.Thus, we hope that the Reviewer will agree that SCs forms a better characterized and ultimately more homogeneous group of control than would even age-and gender-matched "healthy controls".
-The differences in the levels of CBX5 detected by RNA-seq need to be independently confirmed by qRT-PCR and western blotting.The entirety of the original monocyte preparations was lyzed and all the yielded RNA was used for the preparation of the libraries used in the RNA-seq reactions.Thus, we can provide neither Westernblots, nor RT-qPCR reactions on these samples.Yet, we still had available the fractions complementary to the monocytes after the Miltenyi columns, enriched in T and B cells.PCR reactions on these samples has allowed to confirm the variations in CBX5 gene expression between the groups.As will be further described below, these leftover samples have also allowed us to document the defective pause-release in Lo-CBX5 patients, using cells other than monocytes.These data are now presented in new Figures 3E-3H and Sup.Figures 3B-3E.
-The plot of EAE clinical scores in Fig. 4B lacks the necessary statistics to confirm the significance of the differences between genotypes.We have now clearly indicated that the error bars represent standard error of the mean (SEM), which was indeed missing.This was also corrected for panels Sup.4A and Sup.4B.
-The characterization of the EAE model is largely incomplete.The analysis of EAE scores should be complemented with histological evaluation of immune cell infiltration and demyelination in the spinal cord.In addition, the peripheral autoimmune response should be assessed in terms of pro-inflammatory vs tolerogenic mediators (Th1/Th17 T cells, cytokines and so on).We recognize that a more thorough characterization of the EAE model is necessary for a comprehensive understanding of the impact of Cbx5 inactivation on mouse immunity.Unfortunately, none of the current co-authors have sufficient know-how in immunology to carry out this characterization.Thus, to carry out these experiments, we have teamed up with a group in Toulouse and are requesting funding for these experiments.However, these experiments will require importing the mice to Toulouse, which would not be compatible with the 3-month period allowed for the revision, and we hope the Reviewer agrees that this work should be included in a new manuscript with a different set of authors.
-It is not clear the number of mice that was used for the EAE experiment for each genotype.Same for the number of independent immunizations that were carried out.Given the high degree of variability of the model and the relatively small differences in clinical scores between genotypes, multiple immunizations are required to confirm the results.EAE was carried out by immunizing 10 WT mice, 10 Cbx5+/-mice, 6 Cbx5 -/-mice (more difficult to breed).As one of Cbx5-/-mice died at day 12, presented results only include data from 5 of the KO mice.The RNA-seq data was carried out on a second set of mice including 2 WT, 1 Cbx5+/-, and 3 Cbx5-/-mice used as controls, and 3 WT and 3 Cbx5-/-mice exposed to an EAE protocol arrested at day 14.This is now clearly indicated in the Material and Method section of the manuscript.In addition, we have included in Sup. Figure 4B, the tacking of the body weight of the Cbx5-/-mice, which provides a quantitative complement to the clinical scores.
-It is not clear the rationale for performing RNA-seq on CD4+ T cells in EAE mice while monocytes were investigated in MS patients.The analysis in EAE mice should be extended to monocytes for consistency.Repeating the RNA-seq analysis on monocytes from Cbx5 KO mice would not have been possible in the 3-month period allowed for the revision.However, to improve the consistency of the manuscript, we have (1) leveraged the complementary fractions from our monocyte purification, enriched in B and T cells, to examine manifestations of RNAPII pausing in non-monocyte cells at a couple of representative genes.This approach shows that patients accumulating reads on the 5' region of genes in monocytes also yield a greater RT-PCR signal on this region (as compared to more 3' regions) in the PBMCs leftover from the monocyte purification.These data are now presented in new Figure 3E-3H and Sup. Figure 3C-3E.In addition, we have examined publicly available transcriptomic data from MS patient CD4+ T cells (Kim et al. 2020 -New Ref #22).Our analysis shows that by segregating patients based on CBX5 expression levels, genes with significantly altered expression between high and low CBX5 groups are enriched in NELFE targets, similar to our observations in monocytes.These findings strongly suggest that in T cells, as in monocytes, reduced levels of CBX5 are primarily associated with the deregulation of genes highly dependent on promoter escape for their regulation.These results are now presented in Sup.Figures 2A-2D.
-More than 200 loci have been found associated with MS risk in a recent GWAS (IMSGC, Science 2019).It would be relevant to test whether the low-CBX5 group carries any of those risk SNPs in the CBX5 locus.The IMSGC study identifies a risk SNP in INTS8.Therefore, the association of INTS8 with an increased risk of MS can now be supported by two different references (Ref #26, and #27 -IMSGC), and we thank the reviewer for highlighting this.Conversely, CBX5 is, to our knowledge, not associated with an increased risk of MS, including findings from the IMSGC study.It is possible that alterations in the CBX5 gene may be lethal in humans.This point is now addressed in the discussion section.Thank you for submitting your revised manuscript entitled "Defective Integrator activity shapes the transcriptome of patients with multiple sclerosis".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Fig 2B, Fig 5A: Additional Integrator subunits have been identified beyond INTS1-12.These include INTS13, INTS14, INTS15 and PP2A subunits.The authors should analyze their expression levels as well.These subunits have been included in Fig 2B (human) and 5A (mouse).Minor comments: (1) Fig 1B: Words in figure are small and hard to read.The font has been adjusted to match other text in the Figure (Arial 10).(2) Fig 1F: Please explain the figure labels more so that a reader can more easily understand how this figure shows an enrichment for Vitamin D regulated genes as described in the text.We now explain in the Figure legend that VDR stands for vitamin D receptor.(3) Line 205: It could be interesting that INTS6 is up-regulated as recent work suggests INTS6 over expression can block Integrator activity.PMID: 37995689 This reference is now cited (Reference #40) and we thank the reviewer for the suggestion.(4)Line 209: The authors should cite PMID:16239144 as this was the earliest report of Integrator acting on snRNAs.This reference is now cited (Reference #28).
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Thank you for submitting your Research Article entitled "Defective Integrator activity shapes the transcriptome of patients with multiple sclerosis".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.